Project Summary/Abstract The dental pulp is composed of a supporting reticular matrix containing blood vessels, nerves, stromal fibroblasts and few specific stem cells. Regenerative medicine has focused on the dental pulp stem cell as a ready source of multipotential adult stem cells. However, the dental pulp fibroblast also may provide important cues for the functioning dental pulp. Included is the dental pulp fibroblast?s support of nerve repair. This project adopts an innovative and alternative approach to regenerative sciences by leveraging the neurotrophic features of dental pulp fibroblasts, potentially for nerve regeneration therapies. Here we explore ways of enhancing this neurotropic function of pulp fibroblasts via a novel C5L2 pathway involving p38 map kinase (p38) signaling. We recently studied the complement C5a receptor (C5aR) as one of the initial events that control the brain-derived neurotrophic factor (BDNF) secretion. This resultant BDNF secretion induces neurite outgrowth towards the injury site. Whereas C5a is known to interact with its G-coupled protein receptor C5aR, another controversial C5a receptor has been cloned. The C5L2 was considered as a non-functional decoy receptor of C5a signaling, thus has received much less attention than C5aR. Preliminary data demonstrate that C5L2 inhibition by siRNA significantly increases the pulp fibroblast-mediated BDNF secretion and neurite outgrowth. We provide further evidence that p38 plays a key role in the pulp fibroblast-mediated BDNF modulation. Based on our observations, we hypothesize that pulp fibroblasts enhance nerve regeneration by a paracrine function of secreted neurotrophic factors via the C5L2 and p38 pathways. In this proposal, we will characterize the mechanisms of C5L2 action in the pulp fibroblast-mediated nerve outgrowth in vitro. We will use an axon investigation system device (AXIS) to identify the interaction with C5L2 and p38, and whether C5L2 function is dependent or independent of C5a/C5aR. We will further determine, in vivo, whether C5L2 acts on pulp fibroblast functions to control IAN regeneration using our mouse IAN regeneration model by transplantation of the C5L2-silenced pulp fibroblasts. In our follow-on R01, we will investigate how pulp fibroblast-mediated nerve regeneration and C5L2 signaling impact functional plasticity: (a) whether the gene knockout approaches demonstrate nerve regenerative potential using in vivo IAN regeneration model, (b) whether the transplantation of engineered pulp fibroblasts and dental pulp stem cells that constitutively express and BDNF into the IAN denervation model can enhance nerve regeneration, (c) the consequence of nerve regeneration regarding pulp and dentin healing, and (d) the consequence of nerve regeneration regarding a behavioral (i.e., pain) effect. The results obtained from this project will shed new light onto cellular and molecular events which orchestrate initial steps of nerve regeneration by linking the neurite outgrowth to pulp fibroblast function through C5L2 and p38 pathways. These studies will provide the basis for future potential therapeutic interventions of nerve repair and vital tooth preservation.